IMPROVED MUTATION ANALYSIS OF THE NF1 GENE

摘要

The present invention relates to a method for mutation analysis of the neurofibromatosis 1 (NF1) gene of a patient comprising the steps of extracting DNA from peripheral blood lymphocytes of said patient, establishing an EBV transformed B-lymphoblastoid cell line using lymphocytes from said patient, treatment of said EBV transformed B-lymphoblastoid cell line culture with puromycin, immediate extracting RNA of cultures of said cell line, amplifying said RNA using suitable primers, and, obtaining peptide fragments by means of in vitro transcription/translation of said amplified fragments. This new approach allows a much faster, sensitive and reliable analysis of any NF1 mutation that is normally difficult to perform due to unstable and wrong-spliced transcripts. Using this technique, mutations within the NF1 gene can be detected in 83% of all cases, which is significantly higher compared to previously described analysis techniques (71%), and do not result in false positives which are expectable from other setups. This invention also relates to the identification of new hotspots and specific NF1 mutations. This invention comprises diagnostic kits for the detection of described specific mutations and hotspot domains, compounds correcting the structure of specific mutated NF1 proteins and in vitro and in vivo systems that can be used to screen for these therapeutic compounds.